Mytilus edulis, also termed the common edible mussel or blue mussel, constitutes most of the world's commercial production of cultured mussels, along with the closely related species Mytilus galloprovincialis. Besides their use in food culturing, mussels (which is an example of a molusk) have also been used to monitor pollutants in coastal marine waters. The most extensive research about the adhesive properties of mussels has been with M. edulis. 
Marine mussels, like the edible blue mussel, M. edulis, attach to a variety of surfaces in an aqueous environment using a natural adhesive that is incredibly strong and durable. There are no conventional glues that can be applied in an aqueous environment and are impervious to water and turbulent forces. Prior research has shown that one of the proteins in the adhesive, Mytilus edulis foot protein 1 (Mefp-1), bonds to glass, plastic, wood, concrete and Teflon. Nine other adhesive-related proteins from M. edulis have been identified to date. A tenth is implicated, but has not been identified. The precise mechanism for assembly of the ten proteins is not understood (Mefp-1, -2, -3, -4, -5; Collagens: Precollagen-D, —P (variant P22 and P33), Precollagen-NG, Proximal Matrix Thread Protein (1 and 1a); catechol oxidase). There also may be additional proteins involved in the formation of the adhesive.
Individual protein components have been previously identified from byssal structures through protein isolation and amino acid analysis, revealing repetitive amino acid motifs and modified amino acids with unique characteristics not found in other biological systems. Proposed mechanisms for the strength and waterproof properties of the adhesive formed, relate to these recurring amino acid motifs and hydroxylated amino acids found in many of the protein components. Commercial recombinant protein products consisting of either the partial amino acid sequence of Mefp-1 or repeats of the unique decapeptide motif have been marketed in the past. However, no commercial product incorporates any of the other proteins known to be involved in underwater adhesion by the M. edulis mussel. Furthermore, these products are a result of protein isolation techniques and NOT recombinant DNA techniques.
Initial strategies for identifying the adhesive proteins of the byssus of M. edulis involved purification of the proteins directly from the byssi of thousands of animals. About 10,000 mussels are needed to produce 1 gram of adhesive. Thus, subsequent purification and microscopic analysis require(d) the sacrifice of many mussels. This is neither environmentally friendly nor economically practical. When the original mussel adhesive protein, MAP, was identified, only the amino acid motif common to this protein, also referred to as Mefp-1, (a decapeptide repeat occurring ˜80 times) was used in an alternate host production scheme. This MAP recombinant protein did/does have substantial adhesive properties; however, the (complete) gene sequence for Mefp-1 and the other proteins involved in byssus formation are necessary for mimicking the bioadhesive. In addition to a full length Mefp-1, isolating, purifying and sequencing the DNA sequence of M. edulis ' foot protein-2 (Mefp-2) are critically important and are objectives of the present invention.
The mussel byssus is an extracorporeal structure that consists of a stem, thread, and a plaque (also referred to as a pad or disc) (See FIG. 1) This exogenous attachment device was first described in Brown C H, Some Structural Proteins of Mytilus edulis, Quarterly Journal of Microscopical Science, 93(4): 487 (1952). High concentrations of polyphenolic proteins (e.g. L-DOPA), the presence of collagen, and the presence of a catechol oxidase were among the first observations of byssal attachments. Environmental factors such as salinity, temperature, pH, season, and substratum choice, as well as biological factors such as age and metabolic state of the animal effect the efficiency and strength of bonding/attachment. See Crisp D J, Walker G, Young G A, Yule A B, Adhesion and Substrate Choice in Mussels and Barnacles, Journal of Colloid and Interface Science, 104 (1): 40–50 (1985).
The stem is rooted in the byssal retractor muscles at the base of the foot organ. See Crisp D J, Walker G, Young G A, Yule A B, Adhesion and Substrate Choice in Mussels and Barnacles, Journal of Colloid and Interface Science, 104 (1): 40–50 (1985). The byssal threads, flexible structures of variable dimensions (e.g. ˜0.1 mm diameter, 2–4 cm length) and strength, originate from the stem. A byssal thread consists of a flexible, collagenous inner core surrounded by a hard, browned polyphenolic protein. Numerous researchers photographed the collagen core in the 1930's (See Brown C H, Some Structural Proteins of Mytilus edulis, Quarterly Journal of Microscopical Science, 93(4): 487 (1952))—well before three unique, collagenous proteins were identified and characterized by J. H. Waite and colleagues. The outer polyphenolic protein, believed to undergo a curing or quinone tanning-type reaction with a specialized catechol/polyphenol oxidase enzyme, is traditionally designated as Mytilus edulis foot protein 1, Mefp-1, or MAP. (Designation of the byssal thread polyphenolic adhesive protein, as well as subsequent adhesive proteins identified in M. edulis, is preceded by the genus and species: e.g. Mytilus edulis foot protein 1=Mefp-1).
The breaking energy of byssal threads is reported to be 12.50×106 Jm−3, vs tendon (2×106 Jm−3 to 5×106 Jm−3) and silk (50×106 Jm−3 to 180×106 Jm−3; See Denny M W, Biology and the Mechanics of the Wave Swept Environment, Princeton: Princeton University Press (1988); Qin X X, Waite J H, Exotic Collagen Gradients in the Byssus of the Mussel, Mytilus edulis, Journal of Experimental Biology, 198 (3): 633–644 (1995). Bond strengths range from 0.1 to 10×106 Nm−2 depending on the substratum. (See Waite J H, Reverse Engineering of Bioadhesion in Marine Mussels, Bioartificial Organs II: Technology, Medicine, and Materials Annals of the New York Academy of Sciences, 875: 301–309 (1999)). Byssal thread strength at the distal portion of threads is as strong as vertebrate tendon, but 3–5× more extensible (see, Qin X X, Waite J H, A Potential Mediator of Collagenous Block Copolymer Gradients in Mussel Byssal Threads, Proceedings of the National Academy of Sciences of the United States of America, 95 (18):10517–10522 (1998)). Byssal thread strength at the proximal portion of threads is weaker, but 15–20× more extensible. Strain energy density of threads approaches that of silk at approximately 6× tougher than tendon. Byssal threads can recover initial length and stiffness given sufficient relaxation time (See Bell E C, Gosline J M, Mechanical Design of Mussel Byssus: Material Yield Enhances Attachment Strength, Journal of Experimental Biology, 199 (4): 1005–1017 (1996). The byssal structure culminates in a polyphasic plaque of varying size, dependent upon both the size of the animal and the age of the byssus (See Crisp D J, Walker G, Young G A, Yule A B, Adhesion and Substrate Choice in Mussels and Barnacles, Journal of Colloid and Interface Science, 104 (1): 40–50 (1985). Plaques are commonly only ˜0.15 mm in diameter where they meet the thread, and ˜2–3 mm diameter at the substrate interface. Plaque formation occurs from the deposition of proteins that originate from the foot organ. To date, four specialized adhesive proteins have been identified in byssal plaques from M. edulis: Mefp-2, Mefp-3, Mefp-4 and Mefp-5.
In spite of the extensive research in this area, and relative success in patenting and commercializing aspects of these adhesive proteins, a complete understanding of how the byssus is assembled from its component proteins, and the role each protein plays in successful assembly and attachment has not been achieved. A major hurdle has been, and remains, large-scale production of protein in quantities to allow extensive study outside of the byssus. This invention describes nucleotide sequences from cDNAs for Mefp-2 for the first time.